Create Merge Object Cluster and make UMAPS Calculate proportions Label main cell groups Label cell subtypes
Read in the processed data object with merged 4 populations
Check CD24 - fix sample name mix up
Now remove all the predictions and unnecessary clustering
First get a vector of the metadata
meta.to.remove <- meta.v[7:362]
meta.to.remove
[1] "RNA_snn_res.0"
[2] "RNA_snn_res.0.2"
[3] "seurat_clusters"
[4] "RNA_snn_res.1.2"
[5] "percent.rb"
[6] "pANN_0.25_0.09_110"
[7] "DF.classifications_0.25_0.09_110"
[8] "predicted.id"
[9] "prediction.score.RGa"
[10] "prediction.score.Astrocytes.2"
[11] "prediction.score.Neurons.e"
[12] "prediction.score.Oligodendrocytes"
[13] "prediction.score.Neurons.DA"
[14] "prediction.score.Neural.Precursors"
[15] "prediction.score.Neurons.i"
[16] "prediction.score.Neurons"
[17] "prediction.score.Other"
[18] "prediction.score.Epithelial"
[19] "prediction.score.Astrocytes.1"
[20] "prediction.score.RGd1"
[21] "prediction.score.RGd2"
[22] "prediction.score.max"
[23] "MBOAST23.pred"
[24] "prediction.score.Neurons_mature"
[25] "prediction.score.Neurons_DA"
[26] "prediction.score.Neurons_early_inh"
[27] "prediction.score.RGd3"
[28] "prediction.score.epithelial"
[29] "prediction.score.Neurons_early_inh2"
[30] "prediction.score.Neurons_DA_NPC"
[31] "prediction.score.Astrocytes2"
[32] "prediction.score.Astrocytes1"
[33] "prediction.score.oligo"
[34] "prediction.score.Neurons_early2"
[35] "prediction.score.OPC"
[36] "prediction.score.Neurons_early"
[37] "prediction.score.NPC"
[38] "prediction.score.Neurons_inh"
[39] "MBOAIW.pred"
[40] "prediction.score.Dopaminergic.Neurons.early.2"
[41] "prediction.score.Radial.Glia.1"
[42] "prediction.score.Dopaminergic.Neurons.early.1"
[43] "prediction.score.Neural.Epithelial"
[44] "prediction.score.Astrocytes"
[45] "prediction.score.Dopaminergic.Neurons.1"
[46] "prediction.score.Radial.Glia.2"
[47] "prediction.score.Dopaminergic.Neurons.2"
[48] "prediction.score.Dopaminergic.Neurons.3"
[49] "prediction.score.Dopaminergic.Neural.Precursors"
[50] "AIW60.pred"
[51] "RNA_snn_res.0.6"
[52] "subgroups"
[53] "prediction.score.Dividing_5"
[54] "prediction.score.Dividing_3"
[55] "prediction.score.Neuron_4"
[56] "prediction.score.Neuron_5"
[57] "prediction.score.Dividing_4"
[58] "prediction.score.Neuron_37"
[59] "prediction.score.RG_4"
[60] "prediction.score.Dividing_2"
[61] "prediction.score.RG_45"
[62] "prediction.score.Astrocyte_5"
[63] "prediction.score.Endo_5"
[64] "prediction.score.Endo_15"
[65] "prediction.score.Endo_13"
[66] "prediction.score.Neuron_100"
[67] "prediction.score.Interneuron_24"
[68] "prediction.score.Interneuron_1"
[69] "prediction.score.GW14_Microglia_27"
[70] "prediction.score.Neuron_22"
[71] "prediction.score.Endo_7"
[72] "prediction.score.RG_1"
[73] "prediction.score.Oligo_6"
[74] "prediction.score.Oligo_2"
[75] "prediction.score.Endo_4"
[76] "prediction.score.Neuron_2"
[77] "prediction.score.Astrocyte_4"
[78] "prediction.score.Oligo_11"
[79] "prediction.score.Endo_8"
[80] "prediction.score.Neuron_98"
[81] "prediction.score.Microglia_2"
[82] "prediction.score.RG_6"
[83] "prediction.score.RG_7"
[84] "prediction.score.RG_37"
[85] "prediction.score.Dividing_11"
[86] "prediction.score.Endo_11"
[87] "prediction.score.Microglia_8"
[88] "prediction.score.Oligo_8"
[89] "prediction.score.Microglia_10"
[90] "prediction.score.Dividing_42"
[91] "prediction.score.Interneuron_9"
[92] "Bha.mid.stri.pred"
[93] "prediction.score.dividing"
[94] "prediction.score.neuron"
[95] "prediction.score.radial.glia"
[96] "prediction.score.astrocyte"
[97] "prediction.score.endothelial"
[98] "prediction.score.interneuron"
[99] "prediction.score.microglia"
[100] "prediction.score.oligodendrocyte"
[101] "Cell_Types"
[102] "pANN_0.25_0.09_773"
[103] "DF.classifications_0.25_0.09_773"
[104] "number.groups"
[105] "cellsubgroups"
[106] "pANN_0.25_0.09_3000"
[107] "DF.classifications_0.25_0.09_3000"
[108] "prediction.score.Astro_VIM_TNFSRF12A"
[109] "prediction.score.Astro_GLYATL2"
[110] "prediction.score.Astro_GUCY1A2"
[111] "prediction.score.Astro_CYP4F12"
[112] "prediction.score.Astro_GJB6_OXTR"
[113] "prediction.score.Astro_GBP2_SPOCD1"
[114] "prediction.score.Astro_SERPINA3"
[115] "prediction.score.Astro_SIDT1"
[116] "prediction.score.Ependyma_ZBBX"
[117] "prediction.score.Astro_VIM_LHX2"
[118] "astro.pred"
[119] "astro.pred.thresh"
[120] "tree.ident"
[121] "Cell_types"
[122] "prediction.score.RG_3"
[123] "prediction.score.RG_47"
[124] "prediction.score.RG_8"
[125] "prediction.score.Interneuron_3"
[126] "prediction.score.Dividing_13"
[127] "prediction.score.Endo_6"
[128] "prediction.score.GW17_Vascular_14"
[129] "prediction.score.Dividing_7"
[130] "prediction.score.Dividing_48"
[131] "prediction.score.Endo_16"
[132] "prediction.score.RG_46"
[133] "prediction.score.RG_15"
[134] "prediction.score.Neuron_27"
[135] "prediction.score.RG_13"
[136] "prediction.score.Neuron_24"
[137] "prediction.score.Neuron_53"
[138] "prediction.score.Dividing_18"
[139] "prediction.score.GW17_Vascular_21"
[140] "prediction.score.Neuron_26"
[141] "prediction.score.Neuron_102"
[142] "prediction.score.Neuron_10"
[143] "prediction.score.Neuron_36"
[144] "prediction.score.Dividing_12"
[145] "prediction.score.Interneuron_7"
[146] "prediction.score.Dividing_15"
[147] "prediction.score.RG_22"
[148] "prediction.score.Dividing_6"
[149] "prediction.score.RG_11"
[150] "prediction.score.Microglia_3"
[151] "prediction.score.Interneuron_43"
[152] "prediction.score.Dividing_9"
[153] "prediction.score.GW18_Astrocyte_17"
[154] "prediction.score.Neuron_15"
[155] "prediction.score.IPC_18"
[156] "prediction.score.Neuron_101"
[157] "prediction.score.Neuron_18"
[158] "prediction.score.Interneuron_5"
[159] "prediction.score.Neuron_16"
[160] "prediction.score.Neuron_72"
[161] "prediction.score.RG_2"
[162] "prediction.score.Neuron_13"
[163] "prediction.score.Neuron_12"
[164] "prediction.score.GW18_Astrocyte_26"
[165] "prediction.score.Oligo_12"
[166] "prediction.score.Neuron_29"
[167] "prediction.score.GW18_Astrocyte_51"
[168] "prediction.score.Oligo_9"
[169] "prediction.score.Neuron_3"
[170] "prediction.score.Oligo_7"
[171] "prediction.score.Endo_10"
[172] "prediction.score.Neuron_33"
[173] "prediction.score.Dividing_1"
[174] "prediction.score.Astrocyte_2"
[175] "prediction.score.RG_52"
[176] "prediction.score.Neuron_92"
[177] "prediction.score.Neuron_31"
[178] "prediction.score.IPC_34"
[179] "prediction.score.RG_10"
[180] "prediction.score.Dividing_21"
[181] "prediction.score.Neuron_32"
[182] "prediction.score.Neuron_34"
[183] "prediction.score.Endo_9"
[184] "prediction.score.GW18_2_45Oligo"
[185] "prediction.score.Neuron_28"
[186] "prediction.score.Interneuron_6"
[187] "prediction.score.Endo_1"
[188] "prediction.score.GW18_2_42Neuron"
[189] "prediction.score.Astrocyte_1"
[190] "prediction.score.Microglia_6"
[191] "prediction.score.Neuron_93"
[192] "prediction.score.Neuron_39"
[193] "prediction.score.Dividing_10"
[194] "prediction.score.Neuron_6"
[195] "prediction.score.Neuron_35"
[196] "prediction.score.RG_12"
[197] "prediction.score.Dividing_14"
[198] "prediction.score.IPC_29"
[199] "prediction.score.Neuron_19"
[200] "prediction.score.GW19_Dividing_39"
[201] "prediction.score.Interneuron_4"
[202] "prediction.score.GW19_Astrocyte_35"
[203] "prediction.score.RG_17"
[204] "prediction.score.Microglia_5"
[205] "prediction.score.Endo_12"
[206] "prediction.score.RG_59"
[207] "prediction.score.Neuron_50"
[208] "prediction.score.Neuron_1"
[209] "prediction.score.Neuron_17"
[210] "prediction.score.GW19_Astrocyte_53"
[211] "prediction.score.Neuron_11"
[212] "prediction.score.GW19_2_45Outlier"
[213] "prediction.score.Endo_14"
[214] "prediction.score.Microglia_4"
[215] "prediction.score.Oligo_4"
[216] "prediction.score.Oligo_1"
[217] "prediction.score.GW19_2_29Neuron"
[218] "prediction.score.GW19_2_30RG"
[219] "prediction.score.Endo_34"
[220] "prediction.score.Interneuron_31"
[221] "prediction.score.Oligo_3"
[222] "prediction.score.Neuron_8"
[223] "prediction.score.RG_50"
[224] "prediction.score.Neuron_21"
[225] "prediction.score.Interneuron_10"
[226] "prediction.score.GW20_Interneuron_32"
[227] "prediction.score.GW20_Interneuron_34"
[228] "prediction.score.GW20_Astrocyte_39"
[229] "prediction.score.Interneuron_2"
[230] "prediction.score.Astrocyte_6"
[231] "prediction.score.Neuron_7"
[232] "prediction.score.Microglia_15"
[233] "prediction.score.Endo_3"
[234] "prediction.score.Interneuron_8"
[235] "prediction.score.Astrocyte_3"
[236] "prediction.score.RG_9"
[237] "prediction.score.Neuron_68"
[238] "prediction.score.GW22both_RG_38"
[239] "prediction.score.RG_5"
[240] "prediction.score.GW22both_RG_23"
[241] "prediction.score.Microglia_16"
[242] "prediction.score.Microglia_1"
[243] "prediction.score.Oligo_5"
[244] "prediction.score.Dividing_41"
[245] "prediction.score.Endo_2"
[246] "prediction.score.Microglia_76"
[247] "prediction.score.Endo_26"
[248] "prediction.score.Neuron_20"
[249] "prediction.score.Neuron_63"
[250] "prediction.score.Neuron_64"
[251] "prediction.score.intermediate.progenitor.cell"
[252] "prediction.score.outlier"
[253] "Bha"
[254] "pANN_0.25_0.09_604"
[255] "DF.classifications_0.25_0.09_604"
[256] "MBOAST23.thresh"
[257] "AIW120.pred"
[258] "AIW120.thresh"
[259] "AIW60.thresh"
[260] "prediction.score.CALB1_PPP1R17"
[261] "prediction.score.SOX6_AGTR1"
[262] "prediction.score.SOX6_GFRA2"
[263] "prediction.score.SOX6_DDT"
[264] "prediction.score.CALB1_RBP4"
[265] "prediction.score.CALB1_CALCR"
[266] "prediction.score.SOX6_PART1"
[267] "prediction.score.CALB1_GEM"
[268] "prediction.score.CALB1_TRHR"
[269] "prediction.score.CALB1_CRYM_CCDC68"
[270] "da.pred"
[271] "da.pred.thresh"
[272] "prediction.score.Glyc"
[273] "prediction.score.tRG"
[274] "prediction.score.RG.div1"
[275] "prediction.score.nEN.early2"
[276] "prediction.score.nEN.late"
[277] "prediction.score.EN.V1.2"
[278] "prediction.score.vRG"
[279] "prediction.score.RG.div2"
[280] "prediction.score.IN.CTX.CGE1"
[281] "prediction.score.IN.CTX.CGE2"
[282] "prediction.score.IPC.div2"
[283] "prediction.score.oRG"
[284] "prediction.score.IPC.nEN1"
[285] "prediction.score.IPC.div1"
[286] "prediction.score.IN.CTX.MGE2"
[287] "prediction.score.Mural"
[288] "prediction.score.IPC.nEN3"
[289] "prediction.score.IN.CTX.MGE1"
[290] "prediction.score.IPC.nEN2"
[291] "prediction.score.EN.V1.3"
[292] "prediction.score.IN.STR"
[293] "prediction.score.V23"
[294] "prediction.score.EN.PFC3"
[295] "prediction.score.MGE.RG2"
[296] "prediction.score.nIN5"
[297] "prediction.score.MGE.IPC3"
[298] "prediction.score.nIN4"
[299] "prediction.score.MGE.IPC1"
[300] "prediction.score.MGE.RG1"
[301] "prediction.score.MGE.div"
[302] "prediction.score.MGE.IPC2"
[303] "prediction.score.nIN2"
[304] "prediction.score.Astrocyte"
[305] "prediction.score.nIN1"
[306] "prediction.score.EN.V1.1"
[307] "prediction.score.nIN3"
[308] "prediction.score.EN.PFC1"
[309] "prediction.score.nEN.early1"
[310] "prediction.score.EN.PFC2"
[311] "prediction.score.RG.early"
[312] "prediction.score.Endothelial"
[313] "prediction.score.Microglia"
[314] "prediction.score.Choroid"
[315] "prediction.score.U1"
[316] "prediction.score.U2"
[317] "prediction.score.U4"
[318] "prediction.score.U3"
[319] "dev.cortex.pred"
[320] "dev.cortex.pred.thresh"
[321] "dev.cortex.pred.thresh2"
[322] "prediction.score.EarlyExcitatory"
[323] "prediction.score.RadialGlia"
[324] "prediction.score.Glioblast"
[325] "prediction.score.EarlyInhibitory"
[326] "prediction.score.NeuralProgenitor"
[327] "prediction.score.30"
[328] "prediction.score.29"
[329] "prediction.score.VLMC"
[330] "prediction.score.31"
[331] "fb.pred"
[332] "fb.thresh"
[333] "prediction.score.Neuroblast.motorneuron.GABAergic."
[334] "prediction.score.Radial.Glia.cycling"
[335] "prediction.score.Cortical.Interneurons"
[336] "prediction.score.Excitatory.neurons.midbrain"
[337] "prediction.score.Excitatory.neurons.possibly.midbrain"
[338] "prediction.score.OPCs"
[339] "prediction.score.Excitatory.neurons.cortex"
[340] "prediction.score.Forebrain.early.neuroblast.possibly.GABAergic"
[341] "prediction.score.GABAergic.forebrain"
[342] "prediction.score.Cortical.Pyramidal"
[343] "prediction.score.Radial.Glia"
[344] "prediction.score.Radial.Glia.potential.glioblast"
[345] "prediction.score.Radial.Glia.VLMC.primed."
[346] "prediction.score.Glioblast.Pre.OPC"
[347] "prediction.score.Forebrain.neural.progenitor.EMX1"
[348] "prediction.score.Striatum.Cortical.neurons"
[349] "prediction.score.Radial.glia.Glioblast.Forebrain.progenitor.EMX1"
[350] "prediction.score.Forebrain.inhibitory.neuroblast"
[351] "prediction.score.VLMCs"
[352] "prediction.score.Inhibitory.neurons.Midbrain..possibly.GABAergic"
[353] "prediction.score.Hindbrain.neuroblast"
[354] "prediction.score.Radial.Glia.Glioblast"
[355] "prediction.score.GABAergic.or.interneuron.neuroblast.probably.midbrain"
[356] "prediction.score.Midbrain.inhibitory.neuroblast"
Add the subtype labels
# First rename cell subgroup cluster lables
Idents(seu.L) <- 'Level3'
cluster.ids <- c("Astro1","Neurons1","RG1","Astro2","Neurons2",
"Astro3","Astro4","Neurons3","RG2","Neurons4",
"Astro5","Neurons5","RG3","DANeurons1","Neurons6",
"Mix","RG4","NPC","Neurons7","DANeurons2",
"DANeurons3","RG5","RG6","RG7","RG8"
)
# note Neurons8-inh-max is now Neurons 7
# Endothelial NPC is now NPC
# RG6-epi is nw RG6
# RG-OPC is now RG8
# change 'Levels' to 'Level1' main cell types
# keep these as 'Level2'
# later remove 'Level3' - same level just different labels
names(cluster.ids) <- levels(seu.L)
seu.L <- RenameIdents(seu.L, cluster.ids)
seu.L$Level2 <- Idents(seu.L)
DimPlot(seu.L, group.by = 'Level2')
Now look at the previous workbook to make the subgroup labels
Idents(seu.L) <- 'Level2'
cluster.ids <- c("Astrocytes1","Neurons1","RG1","Astrocytes1","Neurons2",
"Astrocytes2","Astrocytes2","Neurons3","RG2","Neurons4",
"Astrocytes3","Neurons5","RG3","DANeurons1","Neurons6",
"Mix","RG4","NPC","Neurons7","DANeurons2",
"DANeurons3","RG5","RG6","RG7","RG8"
)
# note Neurons8-inh-max is now Neurons 7
# Endothelial NPC is now NPC
# RG6-epi is nw RG6
# RG-OPC is now RG8
# change 'Levels' to 'Level1' main cell types
# keep these as 'Level2'
# later remove 'Level3' - same level just different labels
# merge but unmarkered should be Subtypes then I can make Subtype_markers
names(cluster.ids) <- levels(seu.L)
seu.L <- RenameIdents(seu.L, cluster.ids)
seu.L$Cell_Subtypes <- Idents(seu.L)
DimPlot(seu.L, group.by = 'Cell_Subtypes')
NA
NA
NA
NA
NA
Now look at the astrocyte subgroup group markers
DotPlot(sub.astro, features = c("HPD","COL3A1","DCN"), group.by = 'Cell_Subtypes') + RotatedAxis()
Warning: Scaling data with a low number of groups may produce misleading results
DotPlot(seu.L, features = c("HPD","COL3A1","DCN"), group.by = 'Cell_Subtypes') + RotatedAxis()
Double check for other markers
Idents(sub.astro) <- 'Cell_Subtypes'
astro.markers <- FindAllMarkers(sub.astro, logfc.threshold = 0.5, only.pos = TRUE, test.use = "MAST")
Calculating cluster Astrocytes1
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Done!
Calculating cluster Astrocytes2
Completed [=====================================>----------------------] 64% with 0 failures
Completed [========================================>-------------------] 68% with 0 failures
Completed [==========================================>-----------------] 72% with 0 failures
Completed [=============================================>--------------] 76% with 0 failures
Completed [===============================================>------------] 80% with 0 failures
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Completed [====================================================>-------] 88% with 0 failures
Completed [======================================================>-----] 92% with 0 failures
Completed [=========================================================>--] 96% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=====================================>----------------------] 64% with 0 failures
Completed [========================================>-------------------] 68% with 0 failures
Completed [==========================================>-----------------] 72% with 0 failures
Completed [=============================================>--------------] 76% with 0 failures
Completed [===============================================>------------] 80% with 0 failures
Completed [=================================================>----------] 84% with 0 failures
Completed [====================================================>-------] 88% with 0 failures
Completed [======================================================>-----] 92% with 0 failures
Completed [=========================================================>--] 96% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Calculating cluster Astrocytes3
Completed [=======================>------------------------------------] 40% with 0 failures
Completed [=========================>----------------------------------] 43% with 0 failures
Completed [==========================>---------------------------------] 45% with 0 failures
Completed [============================>-------------------------------] 48% with 0 failures
Completed [=============================>------------------------------] 50% with 0 failures
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Completed [====================================================>-------] 88% with 0 failures
Completed [=====================================================>------] 90% with 0 failures
Completed [=======================================================>----] 93% with 0 failures
Completed [========================================================>---] 95% with 0 failures
Completed [==========================================================>-] 98% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [======================>-------------------------------------] 38% with 0 failures
Completed [=======================>------------------------------------] 40% with 0 failures
Completed [=========================>----------------------------------] 43% with 0 failures
Completed [==========================>---------------------------------] 45% with 0 failures
Completed [============================>-------------------------------] 48% with 0 failures
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Completed [==============================>-----------------------------] 52% with 0 failures
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Completed [====================================================>-------] 88% with 0 failures
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Completed [=======================================================>----] 93% with 0 failures
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Completed [==========================================================>-] 98% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Plot possible markers
DotPlot(sub.astro, features = genes) +RotatedAxis()
Warning: Scaling data with a low number of groups may produce misleading results
DotPlot(seu.L, features = genes, group.by = 'Cell_Subtypes') +RotatedAxis()
DimPlot(sub.astro)
FeaturePlot(sub.astro, features = genes)
Label with the subtype markers
Idents(seu.L) <- 'Level3'
cluster.ids <- c("Astrocytes-HPD","Neurons1","RG1","Astrocytes-HPD","Neurons2",
"Astrocytes-COL3A1","Astrocytes-COL3A1","Neurons3","RG2","Neurons4",
"Astrocytes-FABP5","Neurons5","RG3","DANeurons1","Neurons6",
"Mix","RG4","NPC","Neurons7","DANeurons2",
"DANeurons3","RG5","RG6","RG7","RG8"
)
names(cluster.ids) <- levels(seu.L)
seu.L <- RenameIdents(seu.L, cluster.ids)
seu.L$Cell_Subtype_Markers <- Idents(seu.L)
DimPlot(seu.L, group.by = 'Cell_Subtype_Markers')
Check neuron subgroup markers and annotate
Idents(seu.L) <- 'Cell_Subtypes'
sub.da <- subset(seu.L, idents = c("DANeurons1","DANeurons2","DANeurons3"))
DotPlot(sub.da, features = n.markers) + RotatedAxis()
Warning: Scaling data with a low number of groups may produce misleading results
FeaturePlot(sub.da, features = n.markers)
DimPlot(sub.da)
Idents(seu.L) <- 'Cell_Subtypes'
sub.n <- subset(seu.L, idents = c("Neurons1","Neurons2","Neurons3",
"Neurons4","Neurons5","Neurons6","Neurons7"))
DotPlot(sub.n, features = n.markers) + RotatedAxis()
FeaturePlot(sub.n, features = n.markers )
DimPlot(sub.n)
Repeat cluster markers
neur.markers <- FindAllMarkers(sub.n, logfc.threshold = 0.5, only.pos = TRUE, test.use = "MAST")
Calculating cluster Neurons1
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Combining coefficients and standard errors
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Refitting on reduced model...
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Calculating cluster Neurons2
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Combining coefficients and standard errors
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Calculating cluster Neurons3
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Combining coefficients and standard errors
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Calculating cluster Neurons4
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster Neurons5
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster Neurons6
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster Neurons7
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
da.markers <- FindAllMarkers(sub.da, logfc.threshold = 0.5, only.pos = TRUE, test.use = "MAST")
Calculating cluster DANeurons1
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster DANeurons2
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster DANeurons3
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
all.n.markers <- FindAllMarkers(sub.neur, logfc.threshold = 0.5, only.pos = TRUE, test.use = "MAST")
Calculating cluster Neurons1
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [===========================>--------------------------------] 47% with 0 failures
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Done!
Calculating cluster Neurons2
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=========>--------------------------------------------------] 17% with 0 failures
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Done!
Calculating cluster Neurons3
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [==========>-------------------------------------------------] 18% with 0 failures
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Done!
Calculating cluster Neurons4
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [==================>-----------------------------------------] 31% with 0 failures
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Done!
Calculating cluster Neurons5
Completed [===========>------------------------------------------------] 20% with 0 failures
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Completed [============================================================] 100% with 0 failures
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [==========>-------------------------------------------------] 18% with 0 failures
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Done!
Calculating cluster DANeurons1
Completed [=>----------------------------------------------------------] 3% with 0 failures
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster Neurons6
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=====================>--------------------------------------] 37% with 0 failures
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Done!
Calculating cluster Neurons7
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=====>------------------------------------------------------] 10% with 0 failures
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Done!
Calculating cluster DANeurons2
Completed [====>-------------------------------------------------------] 9% with 0 failures
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Done!
Combining coefficients and standard errors
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Refitting on reduced model...
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Calculating cluster DANeurons3
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Done!
Combining coefficients and standard errors
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Done!
Look at the markers in all markers
Rename the neurons with Subtype markers
Now get subtype markers for RG
RG1 - ventral (doesn’t have positive markers) RG2 -
Merge 2 and 4 then get markers again After merging 2 and 4 the new 4 previously 5 also seems the same
Idents(seu.L) <- 'Level3'
DimPlot(seu.L)
cluster.ids <- c("Astrocytes1","Neurons1","RadialGlia1","Astrocytes1","Neurons2",
"Astrocytes2","Astrocytes2","Neurons3","RadialGlia2","Neurons4",
"Astrocytes3","Neurons5","RadialGlia3","DANeurons1","Neurons6",
"Mix","RadialGlia2","NPC","Neurons7","DANeurons2",
"DANeurons3","RadialGlia2","RadialGlia4","RadialGlia5","RadialGlia6"
)
# merge RG2 and 4
# new = previous
# RG1 = "RadialGlia1
# RG2 = RG2
# RG3 = RG3
# RG 4 = RG5
# RG 5 = RG6
# RG6 = RG7
# RG7=RG8
# merge 2 4 and 5
names(cluster.ids) <- levels(seu.L)
seu.L <- RenameIdents(seu.L, cluster.ids)
seu.L$Cell_Subtypes <- Idents(seu.L)
DimPlot(seu.L, group.by = 'Cell_Subtypes')
Calculate new markers
rg.markers <- FindAllMarkers(sub.rg, logfc.threshold = 0.5, only.pos = TRUE, test.use = "MAST")
Calculating cluster RadialGlia1
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Done!
Combining coefficients and standard errors
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Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster RadialGlia2
Completed [===============>--------------------------------------------] 27% with 0 failures
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Completed [============================================================] 100% with 0 failures
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=================>------------------------------------------] 29% with 0 failures
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Done!
Calculating cluster RadialGlia3
Completed [=>----------------------------------------------------------] 3% with 0 failures
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Completed [==>---------------------------------------------------------] 4% with 0 failures
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Completed [===========================================================>] 99% with 0 failures
Completed [===========================================================>] 100% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=>----------------------------------------------------------] 3% with 0 failures
Completed [=>----------------------------------------------------------] 4% with 0 failures
Completed [==>---------------------------------------------------------] 4% with 0 failures
Completed [==>---------------------------------------------------------] 5% with 0 failures
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Completed [===============>--------------------------------------------] 26% with 0 failures
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Completed [===============================================>------------] 81% with 0 failures
Completed [================================================>-----------] 81% with 0 failures
Completed [================================================>-----------] 82% with 0 failures
Completed [=================================================>----------] 83% with 0 failures
Completed [=================================================>----------] 84% with 0 failures
Completed [==================================================>---------] 84% with 0 failures
Completed [==================================================>---------] 85% with 0 failures
Completed [==================================================>---------] 86% with 0 failures
Completed [===================================================>--------] 86% with 0 failures
Completed [===================================================>--------] 87% with 0 failures
Completed [====================================================>-------] 88% with 0 failures
Completed [====================================================>-------] 89% with 0 failures
Completed [=====================================================>------] 89% with 0 failures
Completed [=====================================================>------] 90% with 0 failures
Completed [=====================================================>------] 91% with 0 failures
Completed [======================================================>-----] 91% with 0 failures
Completed [======================================================>-----] 92% with 0 failures
Completed [=======================================================>----] 93% with 0 failures
Completed [=======================================================>----] 94% with 0 failures
Completed [========================================================>---] 94% with 0 failures
Completed [========================================================>---] 95% with 0 failures
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Completed [=========================================================>--] 97% with 0 failures
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Completed [==========================================================>-] 99% with 0 failures
Completed [===========================================================>] 99% with 0 failures
Completed [===========================================================>] 100% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Calculating cluster RadialGlia4
Completed [=========>--------------------------------------------------] 16% with 0 failures
Completed [=========>--------------------------------------------------] 17% with 0 failures
Completed [==========>-------------------------------------------------] 18% with 0 failures
Completed [==========>-------------------------------------------------] 19% with 0 failures
Completed [===========>------------------------------------------------] 19% with 0 failures
Completed [===========>------------------------------------------------] 20% with 0 failures
Completed [============>-----------------------------------------------] 21% with 0 failures
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Completed [=============>----------------------------------------------] 23% with 0 failures
Completed [==============>---------------------------------------------] 24% with 0 failures
Completed [==============>---------------------------------------------] 25% with 0 failures
Completed [==============>---------------------------------------------] 26% with 0 failures
Completed [===============>--------------------------------------------] 27% with 0 failures
Completed [================>-------------------------------------------] 28% with 0 failures
Completed [================>-------------------------------------------] 29% with 0 failures
Completed [=================>------------------------------------------] 30% with 0 failures
Completed [=================>------------------------------------------] 31% with 0 failures
Completed [==================>-----------------------------------------] 31% with 0 failures
Completed [==================>-----------------------------------------] 32% with 0 failures
Completed [===================>----------------------------------------] 33% with 0 failures
Completed [===================>----------------------------------------] 34% with 0 failures
Completed [====================>---------------------------------------] 35% with 0 failures
Completed [=====================>--------------------------------------] 36% with 0 failures
Completed [=====================>--------------------------------------] 37% with 0 failures
Completed [======================>-------------------------------------] 38% with 0 failures
Completed [======================>-------------------------------------] 39% with 0 failures
Completed [=======================>------------------------------------] 40% with 0 failures
Completed [========================>-----------------------------------] 41% with 0 failures
Completed [========================>-----------------------------------] 42% with 0 failures
Completed [=========================>----------------------------------] 43% with 0 failures
Completed [=========================>----------------------------------] 44% with 0 failures
Completed [==========================>---------------------------------] 44% with 0 failures
Completed [==========================>---------------------------------] 45% with 0 failures
Completed [===========================>--------------------------------] 46% with 0 failures
Completed [===========================>--------------------------------] 47% with 0 failures
Completed [============================>-------------------------------] 48% with 0 failures
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Completed [=============================>------------------------------] 50% with 0 failures
Completed [=============================>------------------------------] 51% with 0 failures
Completed [==============================>-----------------------------] 52% with 0 failures
Completed [===============================>----------------------------] 53% with 0 failures
Completed [===============================>----------------------------] 54% with 0 failures
Completed [================================>---------------------------] 55% with 0 failures
Completed [================================>---------------------------] 56% with 0 failures
Completed [=================================>--------------------------] 56% with 0 failures
Completed [=================================>--------------------------] 57% with 0 failures
Completed [==================================>-------------------------] 58% with 0 failures
Completed [==================================>-------------------------] 59% with 0 failures
Completed [===================================>------------------------] 60% with 0 failures
Completed [====================================>-----------------------] 61% with 0 failures
Completed [====================================>-----------------------] 62% with 0 failures
Completed [=====================================>----------------------] 63% with 0 failures
Completed [=====================================>----------------------] 64% with 0 failures
Completed [======================================>---------------------] 65% with 0 failures
Completed [=======================================>--------------------] 66% with 0 failures
Completed [=======================================>--------------------] 67% with 0 failures
Completed [========================================>-------------------] 68% with 0 failures
Completed [========================================>-------------------] 69% with 0 failures
Completed [=========================================>------------------] 69% with 0 failures
Completed [=========================================>------------------] 70% with 0 failures
Completed [==========================================>-----------------] 71% with 0 failures
Completed [==========================================>-----------------] 72% with 0 failures
Completed [===========================================>----------------] 73% with 0 failures
Completed [============================================>---------------] 74% with 0 failures
Completed [============================================>---------------] 75% with 0 failures
Completed [============================================>---------------] 76% with 0 failures
Completed [=============================================>--------------] 77% with 0 failures
Completed [==============================================>-------------] 78% with 0 failures
Completed [==============================================>-------------] 79% with 0 failures
Completed [===============================================>------------] 80% with 0 failures
Completed [===============================================>------------] 81% with 0 failures
Completed [================================================>-----------] 81% with 0 failures
Completed [================================================>-----------] 82% with 0 failures
Completed [=================================================>----------] 83% with 0 failures
Completed [=================================================>----------] 84% with 0 failures
Completed [==================================================>---------] 85% with 0 failures
Completed [===================================================>--------] 86% with 0 failures
Completed [===================================================>--------] 87% with 0 failures
Completed [====================================================>-------] 88% with 0 failures
Completed [====================================================>-------] 89% with 0 failures
Completed [=====================================================>------] 90% with 0 failures
Completed [======================================================>-----] 91% with 0 failures
Completed [======================================================>-----] 92% with 0 failures
Completed [=======================================================>----] 93% with 0 failures
Completed [=======================================================>----] 94% with 0 failures
Completed [========================================================>---] 94% with 0 failures
Completed [========================================================>---] 95% with 0 failures
Completed [=========================================================>--] 96% with 0 failures
Completed [=========================================================>--] 97% with 0 failures
Completed [==========================================================>-] 98% with 0 failures
Completed [===========================================================>] 99% with 0 failures
Completed [============================================================] 100% with 0 failures
Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=======>----------------------------------------------------] 14% with 0 failures
Completed [========>---------------------------------------------------] 15% with 0 failures
Completed [=========>--------------------------------------------------] 16% with 0 failures
Completed [=========>--------------------------------------------------] 17% with 0 failures
Completed [==========>-------------------------------------------------] 18% with 0 failures
Completed [==========>-------------------------------------------------] 19% with 0 failures
Completed [===========>------------------------------------------------] 19% with 0 failures
Completed [===========>------------------------------------------------] 20% with 0 failures
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Completed [==============>---------------------------------------------] 26% with 0 failures
Completed [===============>--------------------------------------------] 27% with 0 failures
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Done!
Calculating cluster RadialGlia5
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster RadialGlia6
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
Completed [=>----------------------------------------------------------] 4% with 0 failures
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Done!
Look at the markers
Check markers
# remember RG1 has no markers
rg <- c("RPL41","PTN","NEAT1","CYP1B1","TOP2A","VCAN")
DotPlot(sub.rg, features = rg) + RotatedAxis()
DotPlot(seu.L, features = rg) + RotatedAxis()
NA
NA
Add the RG cell subtype markers
Remember 2,4 and 5 are merged
Save the new data object
saveRDS(seu.L, paste(pathway, "CombinedLabeledMarkers14102022.RDS"))
Clean up labels
colnames(seu.L@meta.data)
[1] "orig.ident" "nCount_RNA" "nFeature_RNA" "nCount_HTO"
[5] "nFeature_HTO" "percent.mt" "Level3" "Levels"
[9] "Cell_Types" "Level2" "Cell_Subtypes" "Cell_Subtype_Markers"
print("Cell_Subtype_Markers")
[1] "Cell_Subtype_Markers"
unique(seu.L$Cell_Subtype_Markers)
[1] Neurons-GRIA2 NPC Neurons-SPARCL1 DANeurons-TPBG Neurons-CP
[6] DANeurons-RAB3B DANeurons-TTR RadialGlia-CY1B1 RadialGlia-RPL41 RadialGlia-PTN
[11] Astrocytes-FABP5 Astrocytes-HPD RadialGlia-TOP2A Mix Neurons-TPH1
[16] Neurons-TFPI1 Neurons-ASCL1 RadialGlia-NEAT1 RadialGlia-VCAN Neurons-MGP
[21] Astrocytes-COL3A1
21 Levels: Astrocytes-HPD Neurons-ASCL1 RadialGlia-RPL41 Neurons-SPARCL1 ... RadialGlia-VCAN
print("Cell_Subtypes")
[1] "Cell_Subtypes"
unique(seu.L$Cell_Subtypes)
[1] Neurons6 NPC Neurons2 DANeurons2 Neurons4 DANeurons1 DANeurons3
[8] RadialGlia4 RadialGlia1 RadialGlia2 Astrocytes3 Astrocytes1 RadialGlia5 Mix
[15] Neurons7 Neurons3 Neurons1 RadialGlia3 RadialGlia6 Neurons5 Astrocytes2
21 Levels: Astrocytes1 Neurons1 RadialGlia1 Neurons2 Astrocytes2 Neurons3 ... RadialGlia6
print("Cell_Types")
[1] "Cell_Types"
unique(seu.L$Cell_Types)
[1] "Neurons" "Endothelial" "Radial Glia" "Glia" "NPC" "Astrocytes"
[7] "Other"
meta <- colnames(seu.L@meta.data)
meta.to.remove <- c("Level3", "Levels", "Level2")
for(i in meta.to.remove) {
seu.L[[i]] <- NULL
}
colnames(seu.L@meta.data)
[1] "orig.ident" "nCount_RNA" "nFeature_RNA" "nCount_HTO"
[5] "nFeature_HTO" "percent.mt" "Cell_Types" "Cell_Subtypes"
[9] "Cell_Subtype_Markers"